key: cord-0010096-sfhws9td
authors: Melchjorsen, Jesper; Sørensen, Louise N.; Paludan, Søren R.
title: Expression and function of chemokines during viral infections: from molecular mechanisms to in vivo function
date: 2003-06-16
journal: J Leukoc Biol
DOI: 10.1189/jlb.1102577
sha: 646b45e9f90b7e76a848e3d7da0d7ff405f7ad2a
doc_id: 10096
cord_uid: sfhws9td

Recruitment and activation of leukocytes are important for elimination of microbes, including viruses, from infected areas. Chemokines constitute a group of bioactive peptides that regulate leukocyte migration and also contribute to activation of these cells. Chemokines are essential mediators of inflammation and important for control of viral infections. The profile of chemokine expression contributes to shaping the immune response during viral infection, whereas viral subversion of the chemokine system allows the virus to evade antiviral activities of the host. In this review, we discuss the role of chemokines in host‐defense against virus infections, and we also look deeper into the virus‐cell interactions that trigger chemokine expression as well as the cellular signaling cascades involved.

When facing a viral infection, most cell types in the body respond by secreting high levels of type 1 interferons (IFN-␣/ ␤). This induction occurs within a few hours of the infection, and IFN-␣/␤ has a number of antiviral functions [1] . First, IFN-␣/␤ directly induces antiviral activities in the uninfected, neighboring cells. This prevents viral spread by increasing the resistance of uninfected cells toward the virus. Second, IFNs can activate natural killer (NK) cell-mediated cytotoxity toward virus-infected cells [2, 3] . Third, there is accumulating evidence that IFN-␣/␤ contributes to driving the adaptive-immune response in the T helper cell type 1 (Th1) direction via stimulation of IFN-␥ expression [4, 5] .

The cellular antiviral response is initiated by activation of NK cells, which kill virus-infected cells directly and also activate other cells of the innate-and adaptive-immune system through production of cytokines, notably IFN-␥ [6] . One of the IFN-␥-activated leukocytes is the macrophage, which is sensitive to IFN-␥ and other proinflammatory cytokines, including tumor necrosis factor ␣ (TNF-␣) [7] . Macrophages participate in the antiviral response through production of free radicals and proinflammatory cytokines [8] as well as by working as an antigen-presenting cell (APC). It is also possible that macro-phages contribute to antiviral defense via phagocytosis of extracellular virus and infected apoptotic cells [7] . Dendritic cells (DCs) also participate in the antiviral response through several mechanisms [9] . First, DCs are probably the main APC, thus playing a pivotal role in development and maturation of the specific immune response. Second, this cell type participates in coordinating the inflammatory response, through production of cytokines and chemokines (see below). In particular, cells of the DC lineage have recently been shown to represent the main source of IFN-␣/␤ during viral infections [10, 11] .

Another prominent response to viral infection is the expansion and activation of CD4ϩ and CD8ϩ T cells, which have central roles in antiviral immunity and work through various mechanisms to inhibit replication and clear infection [12] . First, CD8ϩ cells have a direct effector role through cytotoxic T lymphocyte-mediated lysis. Second, this cell type produces IFN-␥ and TNF-␣, cytokines found to possess a prominent portion of the antiviral function of CD8ϩ cells during some viral infections [13] . Third, CD8ϩ cells play a central role in maintenance of the antiviral response by production of chemokines, which modulate the immune response and attract the appropriate leukocyte subsets to the site of infection [2, 14] . The role of CD4ϩ T cells in antiviral immunity is highly dependent on production of cytokines, notably IFN-␥ [15, 16] , and the cytolytic activity exerted by a subset of CD4ϩ T cells [17] .

Activation, coordination, and regulation of the above-described antiviral response are mediated by complex mechanisms, where cytokines play important roles. Within the large group of cytokines, the subgroup of chemotactic cytokines, chemokines, is now also recognized to participate in defense against viral infections. Moreover, chemokines can in some instances be important mediators of the immunopathology associated with many viral infections. In this review, we focus our attention on chemokines and viral infections.

Their main functions are to attract leukocytes to sites of infection and inflammation and to contribute to the homeostatic circulation of leukocytes through tissue [18] . Chemokines also have the capacity to directly activate leukocytes, e.g., to release granula contents and produce cytokines [19] .

Today, ϳ50 chemokines and 20 chemokine receptors have been identified [19] . Chemokines are 80 -130 amino acid residues in size and have a homologous structure consisting of antiparallel ␤-strands with connecting loops held together by disulfide bonds between cysteine residues. The disulfide bonds keep together two domains in the molecule, which are both important for receptor binding and activation [20] . The aminoterminal cysteine residues also form the basis for the systematic classification of chemokines, based on their numbers and mutual placement. The two major groups are CXC chemokines and CC chemokines, both groups having four cysteine residues. In CXC chemokines, the two first cysteine residues are separated by one amino acid, whereas in CC chemokines, the two cysteine residues lie adjacent. Besides the CC and CXC chemokines, there are two minor groups, CX 3 C and C chemokines, with one and two members, respectively. The CX 3 C chemokine CX3CL1/neurotactin (murine) or fractalkin (human), which is a membrane-bound glycoprotein, has three residues between the two amino-terminal cysteines, and the C chemokines have only one amino-terminal cysteine residue [18] .

Chemokines can also be classified based on their pattern of expression. First, homeostatic chemokines are expressed constitutively and participate in recirculation of leukocytes between tissue and lymphatics and in the traffic of leukocytes within compartments of lymph nodes and thymus [21] . Second, inflammatory chemokines are induced by infection and other proinflammatory stimuli and function as mediators of inflammation by attracting leukocytes to the site of infection.

Recently, the nomenclature of chemokines has been changed to a systematic system. The CC chemokines have been renamed CC chemokine ligand (CCL) 1, 2, 3, etc., and the CXC chemokines, CXCL1, 2, etc. [22] . In this paper, we use the new nomenclature but also provide the traditional name when introducing a new chemokine, as most workers in this field, including ourselves, will need some time to get used to the new nomenclature.

The chemokine receptors are seven-transmembrane-spanning, G-protein-coupled receptors that have a number of conserved motifs. They are classified based on the class of chemokines they bind, e.g., CC chemokine receptors (CCRs) bind CCLs, and CXC chemokine receptors (CXCRs) bind CXCLs. Within the group of inflammatory chemokines, there is a great degree of redundancy; i.e., most receptors bind several chemokines, and most chemokines use more than one receptor. In the group of homing chemokines, conversely, most receptors have only one ligand, although there are exceptions (e.g., CCR7). Chemokine receptors are expressed primarily on leukocytes but also on other cells, e.g., endothelial cells. The specific expression of chemokine receptors is dependent on cell type as well as the state of differentiation and activation of the cell [23] .

Besides these receptors, chemokines can bind to nonsignaling molecules. First, in the tissue, chemokines can bind to heparan sulfate proteoglycans in the extracellular matrix or on the surface of cells. As the chemokines retain their chemotactic activity during this binding, they establish a concentration gradient contributing to the attraction of leukocytes [18] . Second, chemokines can bind to the Duffy antigen receptor for chemokines (DARC), expressed on erythrocytes and endothelial cells. DARC can bind CC as well as CXC chemokines and may function as a sink, clearing chemokines from the circulation [18] . A third chemokine-binding protein is D6, which binds CC chemokines but not C, CXC, or CX 3 C chemokines [24] . The function of this receptor, which is expressed on many cell types within and without the hemopoietic system, remains to be understood in detail.

In this review, we discuss what is currently known about chemokines in antiviral defense and review the literature on molecular mechanisms regulating chemokine expression during virus infections. As this review focuses on chemokines in antiviral defense, other aspects of chemokine biology, although important, are not described here. These include the ability of chemokine receptors to function as coreceptors for human immunodeficiency virus (HIV), which is excellently reviewed elsewhere [25, 26] .

To understand the mechanisms governing recruitment of leukocytes to infected areas, knowledge about the chemokine expression profile during viral infections is important. Although most of this work has been done in vitro, a substantial body of information about chemokine expression in vivo is now available. Table 1 summarizes the data on chemokine expression during viral infection in humans as well as in experimental animals. From the available data, it appears that expression of CC chemokines dominates over the expression of CXC chemokines during viral infections. Among the CC chemokines, CCL3/macrophage inflammatory protein (MIP)-1␣ and CCL5/ regulated upon activation, normal T cell-expressed and secreted (RANTES) seem to be almost invariably associated with viral infections, with some interesting exceptions. For instance, during an intranasal influenza virus infection in mice, only CCL2/monocyte chemotactic protein-1 and CXCL10/IFN-␥inducible protein of 10 kDa (IP-10) were detected in the lungs at various time-points post-infection, whereas other chemokines including CCL3 and CCL5 were not expressed [36] . By comparison, RSV-infected mice display high levels of expression of numerous chemokines in the lungs, including CCL3 and CCL5 [38] . This observation in mice correlates very well with the results of a comparative study in pediatric patients infected with RSV and influenza virus [45] . Although serum levels of CCL5 were very high in RSV-infected children, this was not the case in influenza virus-infected patients. This difference in chemokine response to these two viruses may be explained by the ability of influenza virus to inhibit various activities of the immune response, in particular the production and activity of IFNs [46] .

Although CC chemokine expression seems to dominate, CXC chemokines are also expressed. Among the CXC chemokines, expression of CXCL10 is found in many viral infections [36, 38, 40, 43] . CXCL10 together with CXCL9/monokine induced by IFN-␥ (MIG) and CXCL11/IFN-inducible T cell ␣ chemoattractant constitute the non-ELR CXC chemokines, which all use CXCR3 as receptor [18] . Given the strong activity of this receptor as mediator of T cell recruitment, expression of the non-ELR CXC chemokines may be central for mounting an efficient antiviral response. This idea is supported by the finding of a statistically significant association between intrahepatic expression of CXCL9 and the inflammatory activity of chronic hepatitis C [35] . However, as will be discussed below, CXCL9-and CXCL10-dependent functions can also lead to excessive immune activation and accompanying immunopathology [47] .

Another interesting observation from the information shown in Table 1 is the expression of CCL5 in areas of latent herpesvirus infections. For instance, CCL5 expression was detectable in HSV-1-infected TG long after the virus had been cleared [30] , and a different study showed that MHV-68 infection strongly induced expression of many chemokines in the lungs [34] . Moreover, although the virus was rapidly cleared from the lungs, CCL5 protein was present up to 1 month after the infection. These findings show that latently infected cells do stimulate the immune system, despite lack of detectable viral replication, and also explains why CD8ϩ T cells are recruited to TGs latently infected with HSV-1 [48] .

Altogether, viral infections are associated with expression of chemokines, and CC chemokines dominate over the other classes. In the section below, we review the literature regarding the role of chemokines in defense and disease during viral infections.

Viruses themselves provide the best indication that chemokines are important for antiviral defense. The existence of more than 30 virally encoded chemokine and chemokine receptor mimics [49] , at least one of which has been demonstrated to contribute to inhibition of the antiviral host response [50] , strongly suggests that chemokines are important for restriction of virus infections.

Although much remains to be learned about the role of chemokines during viral infections in man, some information is available. In one study, it was found that expression of CCL5 and CXCL9 correlated statistically with the inflammatory activity in patients with chronic HCV infection [35] . This observation is interesting in light of a recent publication by the group of Chisari (Ref. 47) , where the authors demonstrated that neutralization of CXCL9 and CXCL10 in the murine model for HBV infection did not prevent antiviral defense but diminished liver pathology [27] . Therefore, certain chemokines, although potentially contributing to antiviral activity, may also fuel the host response responsible for the pathology of chronic viral hepatitis.

Chemokines have also been studied in viral meningitis and encephalitis in humans. Among chemokines detected in patients with viral meningitis, notably CCL2 and CXCL10 were abundantly expressed [51] . CCL2 and CXCL10 were identified in the CSF of 97% and 79%, respectively, of the patients and in concentrations sufficient to mediate chemotaxis of mononuclear cells. In a different study, the expression of chemokines in patients with HSV-1 encephalitis was evaluated and compared with the clinical status of the patients [31] . Although several chemokines were detected in the CSF, CCL2 was particularly interesting, as the authors found a strong, reciprocal correlation between the clinical status of the infection and the levels of CCL2. Together, these findings place CCL2 at a cardinal position with respect to defense against virus infections in the central nervous system. As will be discussed below, this idea is supported by studies performed in mice lacking CCR2, the receptor for CCL2 [41] .

Much of our knowledge about the role of specific chemokines and receptors during various viral infections has been gathered through studies in mice. Experimentally, this has been done using specific gene-deleted mice strains or through blocking specific members of the chemokine system with antibodies. As shown in Table 2 , there is now evidence that several chemokines and receptors contribute to antiviral defense and immunopathology. Selected examples are described in more detail below.

Mice deficient in the chemokine receptor CCR1 show increased viral titers after lower respiratory tract infection with PVM, which was found to correlate with enhanced mortality [52, 53] . Conversely, lack of CCR1 does not affect the ability to clear RSV infections, despite high production of the CCR1 ligand CCL3 [53] . Also, mice deficient in CCL3 exhibit delayed viral clearance when infected with influenza virus, PVM, or MCMV [32, 52, 56] . In an impressive series of studies, Biron and colleagues [32, 33, 61] described the leukocyte-cytokine/ chemokine interactions responsible for the antiviral activity of CCL3 in the liver. They identified IFN-␣/␤ as the main inducer of CCL3 expression, which in turn, triggered recruitment of NK cells. This cell type subsequently produced IFN-␥, which was then responsible for induction of expression of CXCL9, a chemokine mediating chemotaxis toward T cells [32, 33, 61] . Collectively, the studies discussed above indicate that CCR1 and its ligands are necessary to raise a sufficient immune response against many viruses. In other viral infections, however, CCL3 has been found not to be important for viral clearance. For instance, lack of CCL3 does not affect viral titers after intraperitoneal Coxsackie B3 virus infection, intranasal RSV infection, and ocular HSV-1 infection [38, 56, 57] . However, in these cases, CCL3 contributes to the pathology of the infections, as CCL3-deficient mice did not develop myocarditis or keratitis after Coxsackie B3 virus and HSV-1 infections, respectively [56, 57] , and the CCL3-deficient mice also displayed lower pulmonary inflammation after RSV infection as compared with wild-type mice [38] .

In another series of studies, respiratory tract infection with influenza virus in CCR2 Ϫ/Ϫ mice caused a decreased inflammatory response, increased viral titers, but reduced mortality when compared with wild-type mice [36] . A similar outcome was seen in mice deficient in CCR2 and CCL3. CCR5 Ϫ/Ϫ mice, however, showed increased infiltration of macrophages, increased pathological changes in the tissue, enhanced mortality, but unchanged viral titers compared with control mice when facing the same infection [36] . A role for CCR5 in immunopathology has also been found during intracerebral infection with MHV. Glass et al. [55] observed that deletion of CCR5 did not affect viral titers and mortality but did decrease postinfectious demyelination when compared with control mice.

Besides attraction of leukocytes to the site of infection, studies have indicated that chemokines also have a role in the development of a Th1 versus Th2 immune response [62] . In mice immunized with DNA vaccines encoding HSV-2 glycoprotein D, coimmunization with CCL5 or CXCL8/IL-8 enhanced an antigen-specific Th1 CD4ϩ T cell-dependent immune response, which correlated with decreased morbidity and mortality during the infection [63] . Moreover, mice lacking CCR2 display an impaired Th1 cytokine profile in the brain after MHV infection despite higher viral titers [41] . In agreement with this, a study in humans showed that in patients infected with HIV, expression of CCL3, CCL4, and CCL5 correlated with a Th1 type of immune response [64] .

Together, these results indicate that the chemokine system is necessary for control of some viral infections and also seems to contribute to shaping the immune response, which in some cases, may lead to an overwhelming inflammatory response with the poitential to cause tissue damage.

In the section above, we have described what is currently known about the functions of chemokines during virus infections in vivo. As these studies have revealed that chemokines are involved in host-defense and immunopathology, there is increasing interest in the molecular mechanisms that govern chemokine expression in virus-infected cells. From Table 1 and the discussion in the text, it is apparent that among the chemokines produced during viral infection, notably, CCL3, CCL5, and CXCL10 are abundantly expressed. Among these, CCL5 is the most studied with respect to molecular mechanisms governing virus-induced chemokine expression. Therefore, the discussion below focuses mainly on CCL5, but we will provide information on other chemokines when available. First, we review the basic principles in virus-cell interactions and virus-activated cellular signal transduction, and subsequently, we discuss the literature on molecular mechanisms involved in regulation of chemokine expression during virus infection.

Virus infection can lead to activation of cellular signal transduction through a number of mechanisms. These include interactions of viral surface proteins with cellular receptors, accumulation of viral double-stranded RNA (dsRNA) produced during the viral life cycle, overload of the cellular protein synthesis machinery, mitochondrial oxidative stress, and virusencoded signaling molecules [65] . Below these mechanisms are described in brief.

First, activation of cellular signal transduction by viral surface proteins occurs through engagement of cellular receptors, which undergo oligomerization or conformational changes following ligand binding and thus, initiate signaling events [66] . Myocardial pathology 2 Virus titers 7 a Ab, Antibody; IL, interleukin. b Symbols used in the text: 2, Parameter decreased in knockout or antibody-treated mice relative to control mice; 1, parameter increased in knockout or antibody-treated mice relative to control mice; 7, parameter unchanged in knockout or antibody-treated mice relative to control mice. MHV-intracerebral infection [40] CD4 ϩ and CD8 ϩ infiltration 2 IFN-␥ and IFN-␤ expression 2 IL-10 production 1 Delayed virus clearance CXCL9 (Ab) Treatment from day 12

CD4 ϩ and macrophage infiltration 7 CCL5 production 7 Infiltration and demyelination 7 CXCL9, CXCL10

HBV-transgenic mice with hepatic HBV replication [47] Virus titers 7 Infiltration of lymphomononuclear cells 2 Liver disease 2 CXCL10-/-MHV-intracerebral infection [60] CD4 ϩ , CD8 ϩ , and macrophage infiltration 2 IFN-␥, CXCL9, and CXCL11 expression 2 Virus titers 1 (day 12)/delayed clearance Demyelination 2 CXCL10

(Ab) Treatment from day 12

MHV-intracerebral infection [59] CD4 ϩ and macrophage infiltration 2 CCL5 production and IFN-␥ expression 2 Demyelination 2 and initiation of remyelination CXCL10

(Ab)

Lymph node cell chemotaxis 2 (ex vivo)

Second, viral dsRNA is produced during viral replication and is sensed by the cell through at least two mechanisms: the dsRNA-activated protein kinase (PKR) and Toll-like receptor (TLR) 3 [67, 68] . Third, overload of viral proteins in the endoplasmic reticulum as well as virus-induced mitochondrial oxidative stress triggers release of calcium from the organelles, which activate kinase cascades, ultimately leading to expression of proinflammatory genes (ref. [69] ; and T. H. Mogensen et al., J. Immunol. in press). Finally, some viral proteins have the capacity to specifically activate certain signal-transduction pathways in infected cells [70] . Although it may not seem beneficial for the virus to deliberately activate signal transduction that promotes antiviral defense, the virus may profit from this by stimulating chemokine expression and hence promote recruitment of new cells to infect, which may subsequently relocate and hence support viral spread in the body [71] . Additionally, some viruses harbor sequences in their gene promoters that are recognized by transcription factors, specifically activated by viral proteins [72] . In the literature, each of the above mechanisms has been reported to activate signaltransduction pathways necessary for cytokine and chemokine production [73] . To illustrate the above principles, the Epstein-Barr virus (EBV) glycoprotein gp350 induces expression of CXCL8 and CCL3 in human neutrophils [74] . This viral glycoprotein uses the cellular surface protein CD21 as receptor and triggers intracellular signaling [75] . Although neutrophils are not the main target for EBV, this finding does demonstrate that gp350 is capable of inducing chemokine expression. Another EBV protein, latent membrane protein 1, which is produced in certain stages of EBV latency and is required for transformation of infected cells, is also able to induce expression of certain chemokines [76] .

Altogether, viruses infect cells and affect the intracellular milieu in a profound manner. The sections below describe the cellular signal-transduction pathways activated by virus infections and how these contribute to chemokine expression.

Virus-activated signal transduction promotes expression of many proinflammatory proteins, including chemokines. In this section, we describe these pathways, which are depicted in Figure 1 .

NF-B is an important cellular, proinflammatory transcription factor, present in the cytoplasm in a latent form associated with IB [77] . Following the appropriate stimulus, IB is serine-phosphorylated by the IB kinase, ubiquitinated, and eventually degraded by the 26S proteasome. Released from IB, the nuclear localization signal of NF-B is exposed, and the transcription factor migrates to the nucleus and activates transcription. Viruses are able to activate NF-B through many different mechanisms. (For a more detailed description, see ref. [73] .) As will be described below in detail, activation of NF-B is indispensable for expression of certain inflammatory chemokines during viral infections.

The IRF family of transcription factors is involved in many aspects of the immune response [78] . For instance, IRF-1-deficient mice fail to develop NK cells and are hyporesponsive to IFN-␥. With respect to virus-induced signal transduction, IRF-3 and IRF-7 are activated in a virus-specific manner and play crucial roles in clearance of virus infections [79] . Like NF-B, the two IRFs require post-translational modification to become activated. Following infection, IRF-3 and IRF-7 are serine/threonine-phosphosphorylated by a kinase, VAK, allowing the proteins to adapt a conformation compatible with homoor heterodimerization. The dimers migrate to the nucleus and activate transcription. There is evidence that IRF-3 and IRF-7 play important roles in virus-induced expression of the cytokine IFN-␣/␤ [79] as well as CCL5 [80] (see below). Recently, IRF-5 has also been linked to virus-induced production of chemokines [81] .

A third group of transcription factors activated by many viruses includes AP-1 and ATF2/Jun. Activation of these transcription factors is dependent on up-stream kinases of the mitogen-activated protein (MAP) kinase family, e.g., p38 and JNK [82] . The transcription factors are active as dimmers, and Jun family members are part of the ATF2/Jun complex and AP-1. The involvement of AP-1 and ATF2/Jun in virus-induced cytokine and chemokine induction is well documented. For example, HSV-2-induced expression of IL-6 and TNF-␣ production relies on ATF2/Jun [83, 84] , and HIV-1 up-regulates production of CCL2 via AP-1 [85] .

Another transcription factor activated in response to virus infection is NF-AT, which is constitutively present in the cytoplasm of some cell types, notably T cells, in a latent, phosphorylated form. Increasing levels of cytoplasmic calcium activate the calmodulin-dependent phosphatase calcineurin, which activates NF-AT by dephosphorylation [86] .

In addition to the above-described examples, virus infection has been reported to affect other cellular transcription factor activities. These include C/EBPs, selective promoter factor 1 (Sp-1), and CRE-binding protein [87, 88] .

In summary, many signal-transduction pathways are activated in virus-infected cells, and our knowledge about the underlying molecular mechanisms for these activities is increasing. Below, we focus the discussion on CCL5 and to a lesser extent, CCL3, CXCL8, and CXCL10 and go through the current knowledge on the mechanisms of chemokine expression in virus-infected cells.

Many chemokines are induced in response to virus infections (Table 1) , and a number of studies have addressed the underlying mechanisms in detail; it appears that common as well as virus-specific activities are involved ( Table 3) . Expression of most chemokines is regulated primarily at the level of transcription, and their gene promoter regions contain recognition sites for many virus-activated transcription factors. Figure 1 depicts the promoters of CCL3, CCL5, CXCL8, and CXCL10, all of which have been studied in the context of viral infections. As it appears from Figure 1 , these promoters contain common as well as promoter-specific regulatory elements, which explains why many chemokines display partly overlapping, yet distinct, patterns of expression. As will be described below, the cooperation between transcription factors binding these promoter elements is of utmost importance in the regulation of chemokine expression.

For CCL5, most of the characterized promoter elements have been shown to contribute to expression of this CC chemokine during virus infection, with NF-B and IRFs being ascribed particularly important roles. This phenomenon was recently explained mechanistically by the finding that NF-B and IRF-3/7 synergistically promote CCL5 expression in Sendai virusinfected fibroblastic or myeloid cells [96] . Subsequently, this observation has been demonstrated also to apply to HSVinduced CCL5 expression (J. Melchjorsen and S. R. Paludan, J. Gen. Virol. in press). The ability of NF-B to act synergistically with IRFs in stimulation of promoter activation has long been appreciated [103] . The above-described, cooperative action of NF-B and IRF factors bears some resemblance to the mechanisms involved in virus-induced expression of CXCL10.

Although differences between the gene promoters of CCL5 and CXCL10 are noticeable, significant similarities are also apparent (Fig. 1) . Most notably, both promoters contain a functional ISRE and two NF-B sites. In fact, as for CCL5, these elements play important roles in regulation of CXCL10 expression during virus infection as well as in response to cytokines [104 -108] . Analysis of the CXCL10 promoter showed that infection with measles virus, NDV, and Sendai virus as well as treatment with dsRNA lead to activation of the wild-type promoter and that this activation is dependent on the region of the promoter encompassing the ISRE and the distal B site [106, 107] . As these findings were done before the identification of IRF-3 and IRF-7 as the main virus-activated IRFs, it was not demonstrated which IRF family member is responsible for the ISREdependent CXCL10 transcription. However, more recent work has indicated that IRF-3 and IRF-7 are involved, given the correlation between hantavirus-induced activation of these two transcription factors and expression of CXCL10 [105] . In the same study, cooperation between IRFs and NF-B was again reported, and this cooperation was found to be enhanced in magnitude and duration by cotreatment with IFN-␥.

NF-B-dependent pathways also regulate CCL3 [109] , whereas IRFs do not seem to participate, an observation explained by the lack of an ISRE element in the CCL3 promoter. The involvement of NF-B, which is activated through a mechanism independent of virus replication, most likely proceeds through the CD28RE. This element is a variant NF-B site, which requires adjacent promoter elements, such as NF-B, Sp-1, or AP-1, for proper function [110] . The CD28RE of the CCL3 promoter lies in close proximity to an AP-1 site (Fig. 1) .

In addition to IRFs and NF-B, C/EBPs and MAP kinasedependent pathways have been demonstrated to stimulate chemokine expression during viral infections [85, 92, 99] . For instance HIV-1 up-regulates production of CCL2 via AP-1 [85] , and influenza A virus infection of human bronchial epithelial cells leads to expression of CCL5 through a mechanism dependent on p38 and JNK [99] . Given the well-described substrate specificities of these two MAP kinases, this indicates that ATF2/Jun stimulates expression of CCL5 in influenzavirus-infected cells. ATFs and Jun have previously been identified to promote expression of CCL5 in response to RSV infection as well as bacterial lipopolysaccharide (LPS) [92, 111] . However, it should be noted that p38 also regulates expression of CCL5 at the post-transcriptional level in RSVinfected airway epithelial cells [93] . Therefore, the role of p38 in expression of CCL5 remains to be fully characterized.

CXCL8, which potently elicits chemotaxis of neutrophils, is expressed during many virus infections in vitro through an AP-1-dependent mechanism [112] [113] [114] [115] [116] [117] . In most reported cases, this occurs through cooperation with NF-B, and examination of the CXCL8 promoter ( Fig. 1 ) reveals two closely located regulatory elements for these transcription factors. AP-1 can also act together with other transcription factors to induce CXCL8 expression. One study suggested that induction of CXCL8 expression proceeds through an AP-1-dependent mechanism during RSV infection, and AP-1 cooperates with ISRE-binding proteins rather than NF-B [115] .

Altogether, a review of the literature demonstrates that the mechanisms through which viruses stimulate expression of chemokines display common as well as virus-type-specific features. In addition, cooperative action of transcription factors plays central roles in the regulation of chemokine expression. For instance, NF-B can stimulate expression of CCL5 through synergistic action with IRFs and also promote expression of CXCL8 via cooperation with AP-1.

As to the virus-cell interactions responsible for activation of signal transduction, important advances have been made recently. Hiscott and colleagues [95] have studied measles virus and IRF factors and found that the nucleocapsid (N) protein is able to activate IRF-3. The authors showed that overexpression of the N protein led to activation of the IRF-3 kinase, VAK, and subsequent phosphorylation of IRF-3. Moreover, the N protein associated with IRF-3 as well as VAK, thus indicating that IRF-3 or an IRF-3-containing protein complex is able to recognize the N protein and transduce this signal to VAK, which in turn, activates IRF-3. The ability of capsid proteins to activate IRF-3 appears not to be specific to papamyxoviruses, as the capsid protein of the flavivirus West Nile virus also induces expression of CCL5 [118] , thus strongly indicating activation of IRF-3. Moreover, HSV-1 has been shown to activate IRF-3 in different human cell lines through a mechanism dependent on viral entry but independent of de novo transcription of viral or cellular genes [119] , again pointing toward a mechanism involving viral capsid proteins.

Although full insight into the mechanism of IRF-3 activation still remains to be gained, the available data suggest that several mechanisms exist. For instance, IRF-3 was initially shown to be activated in response to dsRNA [120] . Moreover, receptor-mediated activation of IRF-3 has also been demonstrated by the finding that LPS activates IRF-3 through engagement of TLR4 [121] . Even highly related viruses may use different mechanisms to activate IRF-3. Krug and associates [122] have compared the ability of influenza A and B viruses with respect to activation of IRF-3 and found that the ability of influenza A virus to activate IRF-3 is sensitive to UV treatment of the virus, whereas that of influenza B virus is not. Thus, activation of IRFs, which can occur through several different mechanisms in virus-infected cells, stimulates expression of CCL5.

Activation of NF-B is very well studied, and a wealth of virus-induced mechanisms has been demonstrated (ref. [73] for a review). For instance, dsRNA activates NF-B through at least two mechanisms. First, the RNA accumulates intracellularly and activates PKR, which in turn, activates the signal transduction leading to degradation of IB and activation of NF-B [67] . Second, dsRNA, in intracellular compartments or extracellularly after release from lysed cells, binds to TLR3 and thus activates NF-B [68] . Studies from this laboratory have shown that HSV infection of macrophages triggers expression of CCL5 and that this relies on PKR, NF-B, and viral-infected cell protein 0 (ref. [123] ; and J. Melchjorsen and S. R. Paludan, J. Gen. Virol., in press). Subsequent studies have identified the mechanism of NF-B activation by HSV to include mitochondrial oxidative stress and subsequent calcium release from this organelle (Mogensen et al., submitted). The laboratory of Dr. Muruve [90, 104] has studied the virus-cell interactions responsible for NF-B-dependent expression of CCL5 and CXCL10 during adenovirus infection. They found that activation of NF-B was mediated by the viral capsid and was dependent on viral entry. Together, these studies show that many different mechanisms contribute to activation of NF-B during viral infections, an essential factor in virus-induced chemokine expression.

The MAP kinase pathways, which contribute to expression of chemokines in response to some virus infections, have also been studied with respect to the viral mechanism of activation [93, 99] . The picornavirus encephalomyocarditis virus and dsRNA have been demonstrated to activate p38 and JNK through PKR-dependent pathways [124] . Others [125, 126] have subsequently confirmed the involvement of PKR in activation of JNK, whereas some controversy remains with respect to the involvement of PKR in activation of p38 by dsRNA. Virus can also activate p38 through dsRNA-independent mechanisms. For instance, adenovirus has been reported to activate the kinase within 20 min of infection [127] , and Muruve and colleagues showed that activation of p38 and ERK by adenovirus was inhibited by bafilomycin A1 or ammonium chloride, suggesting that endosomal escape triggered activation of the MAP kinase pathways. More research is needed to get a better overview of the mechanisms governing activation of MAP kinase pathways during virus infections.

Analysis of the CCL5 promoter reveals at least two additional transcription factor-binding sites potentially involved in regulation of chemokine expression during viral infection, although no direct evidence has been provided yet. First, the GAS element, known to be highly active in response to IFN-␥ [128] , may be activated by homodimers of STAT1 formed in response to IFN-␣/␤, which has been shown to stimulate expression of several chemokines [104] . Second, NF-AT is activated during infections with human T lymphotropic virus type 1 and Sendai virus and probably many more [129, 130] . The presence of a NF-AT-binding site in the CCL5 promoter therefore opens up the possibility that this transcription factor might also play a role in virus-induced CCL5 expression. At this point, it is appropriate to mention that in addition to direct virus-induced chemokine expression, cytokines expressed during the course of infection contribute substantially to stimulation of chemokine production, notably at later stages of infection after the initial virus-host interaction. Therefore, promoter elements not directly targeted by virus-activated transcription factors may still contribute to chemokine production during infection. One study by Matikainen et al. [97] showed that influenza A virus infection of human monocytes triggered expression of a number of chemokines, some of which (CCL2, CCL7, and CXCL10) were dependent on intermediary IFN-␣/␤ expression. Biron and colleagues [61] also found a role for IFN-␣/␤ in virus-induced chemokine expression in the MCMV model, where the authors demonstrated that expression of CCL3 in the liver was induced by type I IFN.

When chemokines were first discovered, it was found that this class of cytokines is a central regulator of leukocyte migration. More recently, chemokines have been ascribed multiple functions, most notably leukocyte activation. Given these properties, it is of little surprise that chemokines contribute significantly to the ability of the organism to mount a proinflammatory response and fight infections. The latter is underscored by the fact that many viruses encode proteins that antagonize chemokine function.

In this paper, we have reviewed what is currently in the literature about the molecular mechanisms through which different viruses activate chemokine production and also what is known about chemokines in viral infections in vivo. Investigations of the molecular events that occur during the clinch between the virus and the host cell have revealed that a wealth of mechanisms is involved. These include interactions between viral glycoproteins and cellular surface receptors; accumulation of viral dsRNA, which is sensed by the cell by various mechanisms; cellular stress through protein overload of the endoplasmatic reticulum or production of reactive oxygen intermediated from the mitochondria; and intracellular accumulation of viral proteins that interact with cellular signal transduction.

As to the role of chemokines during viral infections in vivo, a clear picture still remains to emerge, given the limited number of chemokine knockout mice analyzed in the context of viral infections. The available data seem to support the conclusion that Th1-promoting chemokine receptors and their ligands support viral clearance and in some cases, immunopathology. However, the great degree of redundancy in the chemokine system makes it difficult to predict the role of one specific chemokine or chemokine receptor during infection and hence, emphasizes the importance of thorough experimentation. With the present focus on chemokine receptor antagonists as potential treatment against HIV infections and chronic inflammatory diseases, it is important to fully understand the role of these receptors in defense against infections. Moreover, in-depth understanding of the chemokine system may in turn allow development of vaccines and immunostimulatory drugs as treatment against virus infections in immunocompromised hosts prone to opportunistic infections. 

How cells respond to interferons

Cytokine-mediated control of viral infections

Role of early cytokines, including ␣ and ␤ interferons (IFN-␣/␤), in innate and adaptive immune responses to viral infections

Critical role for STAT4 activation by type 1 interferons in the interferon-␥ response to viral infection

Cutting edge: a murine, IL-12-independent pathway of IFN-␥ induction by gram-negative bacteria based on STAT4 activation by type I IFN and IL-18 signaling

Natural killer cells in antiviral defence: function and regulation by innate cytokines

Interactions between herpes simplex virus and leukocytes: molecular mechanisms and impact on antiviral defense

Macrophage control of herpes simplex virus type 1 replication in the peripheral nervous system

Dendritic cells and viral immunity: friends or foes?

The mannose receptor mediates induction of IFN-␣ in peripheral blood dendritic cells by enveloped RNA and DNA viruses

Natural interferon ␣/␤-producing cells link innate and adaptive immunity

CD8ϩ T cell effector mechanisms in resistance to infection

Noncytolytic control of viral infections by the innate and adaptive immune response

Cytotoxic T lymphocytes, chemokines and antiviral immunity

Interferon-␥ enhances resolution of herpes simplex virus type 2 infection of the murine genital tract

⌫Interferon-dependent protection of the mouse upper respiratory tract following parenteral immunization with a respiratory syncytial virus G protein fragment

Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. I. Lysis restricted by HLA class II MB and DR antigens

Chemokines-chemotactic cytokines that mediate inflammation

Chemokines: immunology's high impact factors

Human chemokines: an update

Chemokines and cell migration in secondary lymphoid organs

International Union of Immunological Societies/World Health Organization Subcommittee on Chemokine Nomenclature (2001) Chemokine/ chemokine receptor nomenclature

Chemokines and T lymphocytes: more than an attraction

Cloning and characterization of a novel promiscuous human ␤-chemokine receptor D6

Co-receptor use by HIV and inhibition of HIV infection by chemokine receptor ligands

HIV-1 receptors and cell tropism

Role of MIP-2 in neutrophil migration and tissue injury in the herpes simplex virus-1-infected cornea

Chemokine synthesis in the HSV-1-infected cornea and its suppression by interleukin-10

Herpes simplex virus replication-induced expression of chemokines and proinflammatory cytokines in the eye: implications in herpetic stromal keratitis

Persistent cytokine expression in trigeminal ganglion latently infected with herpes simplex virus type 1

Time course of chemokines in the cerebrospinal fluid and serum during herpes simplex type 1 encephalitis

A chemokineto-cytokine-to-chemokine cascade critical in antiviral defense

Early murine cytomegalovirus (MCMV) infection induces liver natural killer (NK) cell inflammation and protection through macrophage inflammatory protein 1␣ (MIP-1␣)-dependent pathways

Elevated chemokine responses are maintained in lungs after clearance of viral infection

Increased expression of T cell chemokines and their receptors in chronic hepatitis C: relationship with the histological activity of liver disease

Contrasting effects of CCR5 and CCR2 deficiency in the pulmonary inflammatory response to influenza A virus

Differential expression of proinflammatory cytokine genes in vivo in response to pathogenic and nonpathogenic pneumovirus infections

Inducible expression of inflammatory chemokines in respiratory syncytial virus-infected mice: role of MIP-1␣ in lung pathology

Macrophage inflammatory protein-1␣ and RANTES are present in nasal secretions during ongoing upper respiratory tract infection

Expression of Mig (monokine induced by interferon-␥) is important in T lymphocyte recruitment and host defense following viral infection of the central nervous system

Lack of CCR2 results in increased mortality and impaired leukocyte activation and trafficking following infection of the central nervous system with a neurotropic coronavirus

Central nervous system chemokine expression during Theiler's virus-induced demyelinating disease

Differences in cytokine and chemokine responses during neurological disease induced by polytropic murine retroviruses Map to separate regions of the viral envelope gene

CCR2ϩ and CCR5ϩ CD8ϩ T cells increase during viral infection and migrate to sites of infection

A comparison of cytokine responses in respiratory syncytial virus and influenza A infections in infants

Inhibition of interferon-mediated antiviral responses by influenza A viruses and other negative-strand RNA viruses

Blocking chemokine responsive to ␥-2/interferon (IFN)-␥ inducible protein and monokine induced by IFN-␥ activity in vivo reduces the pathogenetic but not the antiviral potential of hepatitis B virus-specific cytotoxic T lymphocytes

CD8(ϩ) T cells can block herpes simplex virus type 1 (HSV-1) reactivation from latency in sensory neurons

Viral exploitation and subversion of the immune system through chemokine mimicry

The murine cytomegalovirus chemokine homolog, m131/129, is a determinant of viral pathogenicity

Chemotactic activity on mononuclear cells in the cerebrospinal fluid of patients with viral meningitis is mediated by interferon-␥ inducible protein-10 and monocyte chemotactic protein-1

The chemokine macrophage-inflammatory protein-1 ␣ and its receptor CCR1 control pulmonary inflammation and antiviral host defense in paramyxovirus infection

MIP-1␣ is produced but it does not control pulmonary inflammation in response to respiratory syncytial virus infection in mice

The role of CC chemokine receptor 5 in antiviral immunity

Reduced macrophage infiltration and demyelination in mice lacking the chemokine receptor CCR5 following infection with a neurotropic coronavirus

Requirement of MIP-1 ␣ for an inflammatory response to viral infection

Absence of macrophage inflammatory protein-1␣ prevents the development of blinding herpes stromal keratitis

Chemokine induction and leukocyte trafficking to the lungs during murine ␥herpesvirus 68 (MHV-68) infection

Neutralization of the chemokine CXCL10 reduces inflammatory cell invasion and demyelination and improves neurological function in a viral model of multiple sclerosis

IFN-␥-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking

Type I interferons regulate inflammatory cell trafficking and macrophage inflammatory protein 1␣ delivery to the liver

MIP-1␣, MIP-1␤, RANTES, and ATAC/lymphotactin function together with IFN-␥ as type 1 cytokines

DNA vaccines encoding interleukin-8 and RANTES enhance antigenspecific Th1-type CD4(ϩ) T-cell-mediated protective immunity against herpes simplex virus type 2 in vivo

Expression of macrophage inflammatory protein (MIP)-1␣, MIP-1␤, and RANTES genes in lymph nodes from HIVϩ individuals: correlation with a Th1-type cytokine response

Virus-cell interactions: impact on cytokine production, immune evasion and tumor growth

Activation of the erythropoietin receptor by the gp55-P viral envelope protein is determined by a single amino acid in its transmembrane domain

NF-B activation by double-stranded-RNA-activated protein kinase (PKR) is mediated through NF-B-inducing kinase and IB kinase

Recognition of double-stranded RNA and activation of NF-B by Tolllike receptor 3

The ER-overload response: activation of NF-B

The Epstein-Barr virus transforming protein LMP1 engages signaling proteins for the tumor necrosis factor receptor family

Cytomegalovirus-encoded ␤ chemokine promotes monocyte-associated viremia in the host

An inducible transcription factor activates expression of human immunodeficiency virus in T cells

Molecular pathways in virusinduced cytokine production. Microbiol

EBV induces the production and release of IL-8 and macrophage inflammatory protein-1 ␣ in human neutrophils

Epstein-Barr virus binding to CD21 activates the initial viral promoter via NF-B induction

Induction of interleukin-8 by Epstein-Barr virus latent membrane protein-1 and its correlation to angiogenesis in nasopharyngeal carcinoma

Phosphorylation meets ubiquitination: the control of NF-B activity

IRF family of transcription factors as regulators of host defense

Distinct and essential roles of transcription factors IRF-3 and IRF-7 in response to viruses for IFN-␣/␤ gene induction

Essential role of interferon regulatory factor 3 in direct activation of RANTES chemokine transcription

Multiple regulatory domains of IRF-5 control activation, cellular localization, and induction of chemokines that mediate recruitment of T lymphocytes

MAPK signalling cascades in inflammatory disease

Expression of TNF-␣ by herpes simplex virus-infected macrophages is regulated by a dual mechanism: transcriptional regulation by NF-B and activating transcription factor 2/Jun and translational regulation through the AU-rich region of the 3Ј untranslated region

Requirements for the induction of interleukin-6 by herpes simplex virus-infected leukocytes

The human immunodeficiency virus type 1 Tat protein up-regulates the promoter activity of the ␤-chemokine monocyte chemoattractant protein 1 in the human astrocytoma cell line U-87 MG: role of SP-1, AP-1, and NF-B consensus sites

Generic signals and specific outcomes: signaling through Ca2ϩ, calcineurin, and NF-AT

Hepatitis B virus X protein transactivates human interleukin-8 gene through acting on nuclear factor B and CCAAT/enhancer-binding protein-like cis-elements

HBV X protein alters the DNA binding specificity of CREB and ATF-2 by protein-protein interactions

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Papillomavirus type 16 oncogenes downregulate expression of interferon-responsive genes and upregulate proliferation-associated and NF-B-responsive genes in cervical keratinocytes

Multiple cis regulatory elements control RANTES promoter activity in alveolar epithelial cells infected with respiratory syncytial virus

MAPK activation is involved in posttranscriptional regulation of RSV-induced RANTES gene expression

Respiratory syncytial virus-induced RANTES production from human bronchial epithelial cells is dependent on nuclear factor-B nuclear binding and is inhibited by adenovirus-mediated expression of inhibitor of B ␣

Recognition of the measles virus nucleocapsid as a mechanism of IRF-3 activation

Regulation of RANTES chemokine gene expression requires cooperativity between NF-B and IFN-regulatory factor transcription factors

Influenza A and sendai viruses induce differential chemokine gene expression and transcription factor activation in human macrophages

Mechanisms of murine RANTES chemokine gene induction by Newcastle disease virus

mitogen-activated protein kinase and c-jun-NH2-terminal kinase regulate RANTES production by influenza virus-infected human bronchial epithelial cells

Dengue virus infection of human endothelial cells leads to chemokine production, complement activation, and apoptosis

Hantavirus infection induces the expression of RANTES and IP-10 without causing increased permeability in human lung microvascular endothelial cells

Reovirus triggers cell type-specific proinflammatory responses dependent on the autocrine action of IFN-␤

Synergistic action of pro-inflammatory agents: cellular and molecular aspects

Adenovirus vector-induced expression of the C-X-C chemokine IP-10 is mediated through capsid-dependent activation of NF-B

Hantavirus infection induces the expression of RANTES and IP-10 without causing increased permeability in human lung microvascular endothelial cells

IP-10 gene transcription by virus in astrocytes requires cooperation of ISRE with adjacent B site but not IRF-1 or viral transcription

Induction of IP-10 chemokine promoter by measles virus: comparison with interferon-␥ shows the use of the same response element but with differential DNA-protein binding profiles

The interferon-stimulated response element and a B site mediate synergistic induction of murine IP-10 gene transcription by IFN-␥ and TNF-␣

Activation of NF-B by R5 and X4 human immunodeficiency virus type 1 induces macrophage inflammatory protein 1␣ and tumor necrosis factor ␣ in macrophages

Nuclear factor of activated T cells contributes to the function of the CD28 response region of the granulocyte macrophage-colony stimulating factor promoter

ATF and Jun transcription factors, acting through an Ets/CRE promoter module, mediate lipopolysaccharide inducibility of the chemokine RANTES in monocytic Mono Mac 6 cells

Human T-cell leukemia virus type I Tax transactivates human interleukin 8 gene through acting concurrently on AP-1 and nuclear factor-B-like sites

Human cytomegalovirus induces interleukin-8 production by a human monocytic cell line, THP-1, through acting concurrently on AP-1-and NF-B-binding sites of the interleukin-8 gene

Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor constitutively activates NF-B and induces proinflammatory cytokine and chemokine production via a Cterminal signaling determinant

Requirement of a novel upstream response element in respiratory syncytial virus-induced IL-8 gene expression

Interleukin-8 gene regulation in intestinal epithelial cells infected with rotavirus: role of viral-induced IB kinase activation

Hepatitis C virus nonstructural 5A protein induces interleukin-8, leading to partial inhibition of the interferon-induced antiviral response

Induction of potent Th1-type immune responses from a novel DNA vaccine for West Nile virus New York isolate (WNV-NY1999)

Activation of interferon response factor-3 in human cells infected with herpes simplex virus type 1 or human cytomegalovirus

Characterization of specific DNA-binding factors activated by double-stranded RNA as positive regulators of interferon ␣/␤-stimulated genes

Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes

Human influenza viruses activate an interferon-independent transcription of cellular antiviral genes: outcome with influenza A virus is unique

Herpes simplex virus selectively induces expression of the CC chemokine RANTES/CCL5 in macrophages through a mechanism dependent on PKR and ICP0

The protein kinase PKR is required for p38 MAPK activation and the innate immune response to bacterial endotoxin

JNK2 and IKK␤ are required for activating the innate response to viral infection

Activation of p38 mitogen-activated protein kinase and c-Jun NH(2)-terminal kinase by double-stranded RNA and encephalomyocarditis virus: involvement of RNase L, protein kinase R, and alternative pathways

Activation of p38 and ERK signaling during adenovirus vector cell entry lead to expression of the C-X-C chemokine IP-10

Cellular responses to interferon-␥

Activation of the IL-2 gene promoter by HTLV-I tax involves induction of NF-AT complexes bound to the CD28-responsive element

Stimulus-specific assembly of enhancer complexes on the tumor necrosis factor ␣ gene promoter